The developing chicken embryo has been a subject of study since the development of the methods of scientific inquiry. The earliest known detailed description is credited to Aristotle (384-322 B.C.) and a host of other treatises were presented through the ages culminating perhaps in the classical, elementary, english-language treatise "The Development of the Chick" by Frank R. Lillie in 1908. The gross anatomical and microscopic observations of the avian embryo have been almost universally accepted as model observations for embryonic development in general. Numerous texts exist which carefully outline the stages during the 21 day incubation period of the chicken embryo and perhaps the most comprehensive and widely used of these has been "The Avian Embryo" by Alexis L. Romanoff (1960).
Remarkably, however, even this text mentions nothing of the phenomenon of spontaneous movement of embryos within the egg. It has been noted by anyone who has observed developing embryos under strong light (candling), that they move in irregular jumps or spasms as early as the 6th day of incubation up through hatching. Presumably this movement has been accepted as a simple, common occurrence in embryo development and little empirical work on spontaneous movement has been published in contemporary scientific literature. One study (Bursian, A. V., 1964, Bulletin of Experimental Biology and Medicine {Russian}; Vol/ 58, pp 7-11) demonstrated that embryos removed from the shell and connected to movement sensors, responded to light stimulus and exhibited greater spontaneous movement than when not in the presence of light. It is not clear how this relates to in-ovo development however, and no work could be found which actually answers the question of whether embryos move in response to the candling light or whether the embryo moves continuously and candling only illuminates the movement. The consensus from exhaustive personal communications with poultry and avian scientists is that light does stimulate movement of the developing embryo.
The candling of eggs to detect movement has provided a qualitative mechanism for determining whether an embryo is alive or dead or perhaps even "sick", following inoculation of a variety of avian pathogens. In particular, with one avian virus, Newcastle disease virus (NDV), the rate at which the virus kills the embryo has been shown to be directly related to the virulence of the particular strain of virus (Hanson, R. P. and Brandly, C. A., 1955, Science 122, 156-157). That is, the faster the strain kills the infected embryo, the more likely the virus is to be highly lethal in an infected adult bird. This observation has been adapted to provide a diagnostic application for use in identifying highly virulent isolates from field strains of NDV. The procedure involves a staggered inoculation schedule and requires qualitative human assessment at regular time intervals of whether an embryo is alive or dead, based on its response under candling conditions. An averaged "mean death time", (MDT), is then derived for each viral isolate and the number is a reflection of the isolates virulence.
A slightly different, but related approach has been recently developed for the avian influenza (AI) viruses (Perdue, et. al., 1990, Virus Research 16, 137-152) and it may be possible in the future to use a similar diagnostic protocol for the avian influenza field strains.
The present invention avoids the disadvantages of the prior art and provides very significant improvement over the aforementioned procedures, as it permits:
elimination of human qualitative judgments involved in interpreting observations made during manual candling,
higher sensitivity of measurements than was previously possible,
automation of measurements, analysis and data recording,
more frequent and rapid measurements and data processing than manual procedures,
electronic sensing of embryo movement,
rapid and accurate automated quantification of the amount of embryo movement,
rapid and accurate automated measurement of the frequency of embryo movement,
rapid and accurate automated measurement of the pattern of embryo movement,
non-invasive and nondestructive measurement of embryo movement,
elimination of the need to remove incubating eggs from an incubator to take such measurements and
elimination of the adverse effects of manual handling of the eggs.
These and other objects and advantages of the present invention, which will become readily apparent from the ensuing disclosure, are achieved by:
directing light into and through at least one intact avian egg, wherein the light is of sufficient intensity and duration to stimulate embryo movement, whereby portions of the light exit the at least one intact avian egg,
sensing intensities of the portions of the light exiting each of the at least one intact avian eggs,
determining a ratio of, range of sensed light intensities to average sensed light intensities, for each of the at least one intact avian eggs. The aforementioned ratio being a measure of avian embryo movement.
The present invention has wide applicability, including for example: (1) in poultry disease research, for assessing the effects of various inoculants on avian embryos in intact eggs (the ability to rapidly and automatically determine embryo viability at any reading interval would most certainly open avenues which have not yet been opened for investigators in avian diseases and in poultry science studies, e.g. determination of viral pathogenicity on embryos, measuring the effects on virulence of mixing closely related variants of the same strain of virus which differ in pathogenicity i.e. the biological scenario most likely approximating a natural disease outbreak); (2) in hatcheries for determinations of embryo viability in order to sort eggs containing viable embryos from eggs containing non-viable embryos e.g. as an alternative to incubating all of the eggs without screening which results in the incubation of some non-fertile eggs; (3) for detailed studies of movement in developing embryo (classical embryology studies) e.g. if automated determination of gender of developing embryos based on embryo movement could be accomplished, it would revolutionize the breeder industry and primary breeders which presently have to use specialists to determine the gender of 1 day old chicks (it is believed that range frequency and ratio of light intensity for embryonated eggs may be correlated to gender).
Since the present invention may be automated, when it is used with an egg incubator means (i.e. means for incubating at least one egg) it is not necessary for the eggs be removed from the incubator, or that the incubator be opened, when measurements of embryo movement are taken, as is necessary with manual candling techniques. Analogously, the present invention avoids the necessity to enter (or repeatedly enter) into high containment areas to assess embryo viability. Avoiding the necessity to directly access or manually handle the eggs is highly desirable since it eliminates the potentially disruptive effects caused by manual handling (e.g. a drop in incubator and/or egg temperatures for several minutes during and following candling and potential changes in activity or movement caused by the handling).